This revised R21 project is being submitted in response to PA-06-316 for the development of probiotics as complementary and alternative medical (CAM) approaches that overcome the disadvantages of intrapartum antibiotic use (IAU) for the prevention of neonatal bacterial meningitis (NBM). Probiotics have been successfully used for reducing the duration of diarrhea in newborns and children, and for alleviating symptoms of inflammatory bowel diseases. However, it is unknown whether probiotics are beneficial to the prophylaxis of meningitis. Group B Streptococcus (GBS) and E. coli are the two most common bacterial pathogens causing NBM. Extensive studies demonstrated that intrapartum prophylaxis of GBS carriers and selective administration of antibiotics to neonates decrease newborn GBS infection by as much as 80 to 95%. However, widespread IAU, particularly with broad-spectrum antimicrobial agents, has resulted in an increasing incidence of early onset E. coli infections in low birth weight neonates and a rising frequency of ampicillin-resistant E. coli infections in preterm infants. Meningitis caused by enteric bacteria usually begins with intestinal adhesion and invasion. Bacteria enter the blood system and then cross the blood-brain barrier (BBB) to get access into the central nervous system. In order to dissect the pathogenesis of this disease, we have established both cell culture systems (endothelial and epithelial cells) and neonatal rat model of E. coli meningitis. Several host and pathogen virulence factors have been identified and characterized in these systems. Our preliminary data demonstrated that Lactobacillus GG (LGG) was able to suppress meningitic E. coli K1 invasion and transcytosis across human intestinal epithelial cell in vitro (Caco-2 cell culture model) and in vivo (neonatal rat model of E. coli meningitis). Our hypothesis is that probiotic and commensal bacterial organisms (microbiota) may have important influences on the pathogenesis and prophylaxis of neonatal meningitis. Probiotics suppresses meningitic pathogen adhesion to and invasion of host tissue barriers in vitro and in vivo, which are associated with probiotics-enhanced host tissue barrier functions. Our hypothesis will be tested with two Specific Aims. (1). To examine whether the maternal prophylaxis alone or combined with postnatal prophylaxis using probiotics LGG reduces the genesis of bacteremia and the incidence of neonatal E. coli meningitis in the rat model. (2). To determine how probiotic LGG suppresses meningitic E. coli K1 adhesion to and invasion of the host tissues using in vitro models of the gut barrier (Caco-2 & HT-29/cl.f8). The high morbidity and mortality of neonatal bacterial meningitis (NBM) is due to inadequate knowledge of the pathogenesis and prevention of this disease. This R21 grant application, however, should allow us to set up pilot studies to determine whether the maternal prophylaxis alone or combined with postnatal prophylaxis using probiotics Lactobacillus rhamnosus strain GG (LGG) reduces the rate of neonatal E. coli meningitis in the rat model. It should also allow us to determine how LGG suppresses meningitic E. coli K1 adhesion to and invasion of the host tissues using in vitro models of the gut barrier (Caco-2 & HT-29/cl.f8). The overall aims of this proposal are to examine whether NBM can be prevented by probiotics which have beneficial effects on human health and may overcome the crisis in antibiotic resistance due to widespread use of antibiotics. [unreadable] [unreadable] [unreadable]